Protein phosphorylation is important in many cellular processes, including oncogenic transformation. Opal suppressor tRNAs which have phosphoserine attached to them may insert phosphoserine directly into protein by suppressing termination codons. The structure and function of opal suppressor tRNA genes is being investigated by molecular cloning. The project is divided into three parts: 1) an evolutionary study of the sequence of opal suppressor tRNA genes and their flanking sequences in higher eucaryotes (i.e., human, rabbit, chicken, and Xenopus genomes); 2) in vitro transcription of these genes; and 3) site-specific mutagenesis in the promoter and anticodon regions of these genes. Under 1) above, a chicken opal suppressor gene was previously isolated and sequenced. It is encoded in an 87 base pair segment without intervening sequences and has an unusual 5' promoter regions. Two human opal suppressor genes, one normal and one pseudogene, have been isolated, subcloned, and sequenced; three rabbit genes have been isolated subcloned, and are in the process of being sequenced; a second chicken gene has been isolated, subcloned, and is being sequenced; and a Xenopus gene has been isolated and is being subcloned for sequencing. Under 2) above, a transcription system has been prepared from Xenopus oocyte nuclei which is capable of transcribing the chicken and human genes. Under 3) above, we are synthesizing oligonucleotides 17 bases in length which correspond to the 5' internal promoter and to the anticodon regions except for having a single or double base change. The oligonucleotides will be used to make site-specific mutations in the gene.